Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants.

Two different pathways through which protons access cytochrome c oxidase operate during oxygen reduction from the mitochondrial matrix, or the bacterial cytoplasm. Here, we use electrocatalytic current measurements to follow oxygen reduction coupled to proton uptake in cytochrome c oxidase isolated from Paracoccus denitrificans. Wild type enzyme and site-specific variants with defects in both proton uptake pathways (K354M, D124N and K354M/D124N) were immobilized on gold nanoparticles, and oxygen reduction was probed electrochemically in the presence of varying concentrations of Zn(2+) ions, which are known to inhibit both the entry and the exit proton pathways in the enzyme. Our data suggest that under these conditions substrate protons gain access to the oxygen reduction site via the exit pathway.

Effects of butter naturally enriched with conjugated linoleic acid and vaccenic acid on blood lipids and LDL particle size in growing pigs.

BACKGROUND: Cow milk is a natural source of the cis 9, trans 11 isomer of conjugated linoleic acid (c9,t11-CLA) and trans vaccenic acid (VA). These fatty acids may be considered as functional foods, and the concentration in milk can be increased by e.g. sunflower oil supplementation to the dairy cow feed. The objective of this study was to compare the effects of regular butter with a special butter naturally enriched in c9,t11-CLA and VA on plasma lipids in female growing pigs. The experimental period lasted for three weeks and the two diets provided daily either 5.0 g c9,t11-CLA plus 15.1 g VA or 1.3 g c9,t11-CLA plus 3.6 g VA. RESULTS: The serum concentrations of c9,t11-CLA, VA and alpha-linolenic acid were increased and myristic (14:0) and palmitic acid (16:0) were reduced in the pigs fed the CLA+VA-rich butter-diet compared to regular butter, but no differences in plasma concentrations of triacylglycerol, cholesterol, HDL-cholesterol, LDL-cholesterol, LDL particle size distribution or total cholesterol/HDL cholesterol were observed among the two dietary treatment groups. CONCLUSION: Growing pigs fed diets containing butter naturally enriched in about 20 g c9,t11-CLA plus VA daily for three weeks, had increased serum concentrations of alpha-linolenic acid and decreased myristic and palmitic acid compared to pigs fed regular butter, implying a potential benefit of the CLA+VA butter on serum fatty acid composition. Butter enriched in CLA+VA does not appear to have significant effect on the plasma lipoprotein profile in pigs.

Mitochondrial protein sorting: differentiation of beta-barrel assembly by Tom7-mediated segregation of Mdm10.

The mitochondrial outer membrane contains two distinct machineries for protein import and protein sorting that function in a sequential manner: the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex), which is dedicated to beta-barrel proteins. The SAM(core) complex consists of three subunits, Sam35, Sam37, and Sam50, that can associate with a fourth subunit, the morphology component Mdm10, to form the SAM(holo) complex. Whereas the SAM(core) complex is required for the biogenesis of all beta-barrel proteins, Mdm10 and the SAM(holo) complex play a selective role in beta-barrel biogenesis by promoting assembly of Tom40 but not of porin. We report that Tom7, a conserved subunit of the TOM complex, functions in an antagonistic manner to Mdm10 in biogenesis of Tom40 and porin. We show that Tom7 promotes segregation of Mdm10 from the SAM(holo) complex into a low molecular mass form. Upon deletion of Tom7, the fraction of Mdm10 in the SAM(holo) complex is significantly increased, explaining the opposing functions of Tom7 and Mdm10 in beta-barrel sorting. Thus the role of Tom7 is not limited to the TOM complex. Tom7 functions in mitochondrial protein biogenesis by a new mechanism, segregation of a sorting component, leading to a differentiation of beta-barrel assembly.

Essential role of Isd11 in mitochondrial iron-sulfur cluster synthesis on Isu scaffold proteins.

Mitochondria are indispensable for cell viability; however, major mitochondrial functions including citric acid cycle and oxidative phosphorylation are dispensable. Most known essential mitochondrial proteins are involved in preprotein import and assembly, while the only known essential biosynthetic process performed by mitochondria is the biogenesis of iron-sulfur clusters (ISC). The components of the mitochondrial ISC-assembly machinery are derived from the prokaryotic ISC-assembly machinery. We have identified an essential mitochondrial matrix protein, Isd11 (YER048w-a), that is found in eukaryotes only. Isd11 is required for biogenesis of cellular Fe/S proteins and thus is a novel subunit of the mitochondrial ISC-assembly machinery. It forms a complex with the cysteine desulfurase Nfs1 and is required for formation of an Fe/S cluster on the Isu scaffold proteins. We conclude that Isd11 is an indispensable eukaryotic component of the mitochondrial machinery for biogenesis of Fe/S proteins.

Palm oil versus hydrogenated soybean oil: effects on serum lipids and plasma haemostatic variables.

The purpose of this study was to test if replacement of trans fatty acids by palmitic acid in an experimental margarine results in unfavourable effects on serum lipids and haemostatic factors. We have compared the effects of three different margarines, one based on palm oil (PALM-margarine), one based on partially hydrogenated soybean oil (TRANS- margarine) and one with a high content of polyunsaturated fatty acids (PUFA-margarine), on serum lipids in 27 young women. In nine of the participants fasting levels and diurnal postprandial levels of haemostatic variables on the 3 diets were compared. The sum of 12:0, 14:0, 16:0 provided 11% of energy (E%) in the PALM diet, the same as the sum of 12:0, 14:0, 16:0 and trans fatty acids in the TRANS-diet. Oleic acid provided 10-11E% in all three diets, while PUFA provided 5.7, 5.5 and 10.2 E%, respectively. Total fat provided 30-31% and the test margarines 26% of total energy in all three diets. Each of the diets was consumed for 17 days in a crossover design. There were no significant differences in total cholesterol, LDL-cholesterol and apoB between the TRANS- and the PALM-diet. HDL-cholesterol and apoA-I were significantly higher on the PALM-diet compared to the TRANS-diet while the ratio of LDL- to HDL-cholesterol was lower, although not significantly (P = 0.077) on the PALM-diet. Total cholesterol, LDL-cholesterol and apoB were significantly lower on the PUFA-diet compared to the two other diets. HDL-cholesterol was not different on the PALM- and the PUFA-diet while it was significantly lower on the TRANS-diet compared to the PUFA-diet. Triglycerides and Lp(a) were not different among the three diets. The diurnal postprandial state level of tissue plasminogen activator (t-PA) activity was significantly decreased on the TRANS-diet compared to the PALM-diet. t-PA activity was also decreased on the PUFA-diet compared to PALM-diet although not significantly (P=0.07). There were no significant differences in neither fasting levels or in circadian variation of t-PA antigen, PAI-1 activity, PAI-1 antigen, factor VII coagulant activity or fibrinogen between the three diets. Our results suggest that dietary palm oil may have a more favourable effect on the fibrinolytic system compared to partially hydrogenated soybean oil. We conclude that from a nutritional point of view, palmitic acid from palm oil may be a reasonable alternative to trans fatty acids from partially hydrogenated soybean oil in margarine if the aim is to avoid trans fatty acids. A palm oil based margarine is, however, less favourable than one based on a more polyunsaturated vegetable oil.

A diet rich in phosphatidylethanolamine increases plasma homocysteine in mink: a comparison with a soybean oil diet.

The effects of high dietary levels of phosphatidylethanolamine (PE) on plasma concentrations of homocysteine (tHcy) have not previously been studied. Eighteen mink (Mustela vison) studied were fed one of three diets during a 25 d period in a parallel-group design. The compared diets had 0, 17 and 67 % extracted lipids from natural gas-utilising bacteria (LNGB), which were rich in PE. The group with 0 % LNGB was fed a diet of 100 % soyabean oil (SB diet). Phospholipids are the main lipid components in LNGB and Methylococcus capsulatus is the main bacteria (90 %). The fasting plasma concentration of tHcy was significantly higher when the mink consumed the diet with 67 % LNGB than when they consumed the SB diet (P=0.039). A significantly lower glutathione peroxidase activity was observed in mink consuming the 17 % LNGB diet or the 67 % LNGB diet than was observed in mink fed the SB diet. The lack of significant differences in the level of plasma PE due to the diets indicates that most of the PE from the 67 % LNGB diet was converted to phosphatidylcholine (PC) in the liver. It has previously been hypothesised that phosphatidylethanolamine N-methyltransferase is an important source of tHcy. The present results indicate that plasma tHcy is at least partly regulated by phospholipid methylation from PE to PC. This methylation reaction is a regulator of physiological importance.

A comparison of the effects of cheese and butter on serum lipids, haemostatic variables and homocysteine.

Milk fat contains considerable amounts of saturated fatty acids, known to increase serum cholesterol. Little is known, however, about the relative effect of different milk products on risk factors for CHD. The aim of the present study was to compare the effects of Jarlsberg cheese (a Norwegian variety of Swiss cheese) with butter on serum lipoproteins, haemostatic variables and homocysteine. A controlled dietary study was performed with twenty-two test individuals (nine men and thirteen women) aged 23-54 years. The subjects consumed three isoenergetic test diets, with equal amounts of fat and protein, and containing either cheese (CH diet), butter + calcium caseinate (BC diet) or butter + egg-white protein (BE diet). The study was a randomised cross-over study and the subjects consumed each diet for 3 weeks, with 1 week when they consumed their habitual diet in between. Fasting blood samples were drawn at baseline and at the end of each period. Serum was analysed for lipids and plasma for haemostatic variables and homocysteine. Total cholesterol was significantly lower after the CH diet than after the BC diet (-0.27 mmol/l; P=0.03), while the difference in LDL-cholesterol was found to be below significance level (-0.22 mmol/l; P=0.06). There were no significant differences in HDL-cholesterol, triacylglycerols, apo A-I, apo B or lipoprotein (a), haemostatic variables and homocysteine between the diets. The results indicate that, at equal fat content, cheese may be less cholesterol increasing than butter.

Lipids rich in phosphatidylethanolamine from natural gas-utilizing bacteria reduce plasma cholesterol and classes of phospholipids: a comparison with soybean oil.

We compared the effects of three different high-lipid diets on plasma lipoproteins and phospholipids in mink (Mustela vison). The 18 mink studied were fed one of the three diets during a 25-d period in a parallel group design. The compared diets had 0, 17, and 67% extracted lipids from natural gas-utilizing bacteria (LNGB), which were rich in PE. The group with 0% LNGB was fed a diet for which the lipid content was 100% soybean oil. The total cholesterol, LDL cholesterol, and HDL cholesterol of animals consuming a diet with 67% LNGB (67LNGB-diet), were significantly lowered by 35, 49, and 29%, respectively, and unesterified cholesterol increased by 17% compared with the animals fed a diet of 100% lipids from soybean oil (SB-diet). In addition, the ratio of LDL cholesterol to HDL cholesterol was 27% lower in mink fed the 67LNGB-diet than those fed the SB-diet. When the mink were fed the 67LNGB-diet, plasma PC, total phospholipids, lysoPC, and PI were lowered significantly compared with the mink fed a SB-diet. Plasma total cholesterol was correlated with total phospholipids as well as with PC (R = 0.8, P< 0.001). A significantly higher fecal excretion of unesterified cholesterol, cholesteryl ester, PC, lysoPC, and PE was observed in the 67LNGB-fed mink compared with the SB-fed mink. We conclude that phospholipids from the 67LNGB-diet decreased plasma lipoprotein levels, the LDL/HDL cholesterol ratio, and plasma phospholipid levels, especially lysoPC and PC, compared with the highly unsaturated soybean oil. Our findings indicate that the decrease of plasma cholesterol is mainly caused by a specific mixture of phospholipids containing a high level of PE, and not by the dietary FA composition. The lack of significant differences in the level of plasma PE due to the diets indicates that most of the PE from LNGB has been converted to PC in the liver. Thus, plasma cholesterol may at least be partly regulated by phospholipid methylation from PE to PC in the liver.

A diet rich in coconut oil reduces diurnal postprandial variations in circulating tissue plasminogen activator antigen and fasting lipoprotein (a) compared with a diet rich in unsaturated fat in women.

The effects of high and low fat diets with identical polyunsaturated/saturated fatty acid (P/S) ratios on plasma postprandial levels of some hemostatic variables and on fasting lipoprotein (a) [Lp(a)] are not known. This controlled crossover study compared the effects of a high fat diet [38.4% of energy (E%) from fat; HSAFA-diet, P/S ratio 0.14], a low fat diet (19.7 E% from fat; LSAFA-diet, P/S ratio 0.17), both based on coconut oil, and a diet with a high content of monounsaturated fatty acids (MUFA) and PUFA (38.2 E% from fat; HUFA-diet, P/S ratio 1.9) on diurnal postprandial levels of some hemostatic variables (n = 11) and fasting levels of Lp(a) (n = 25). The postprandial plasma concentration of tissue plasminogen activator antigen (t-PA antigen) was decreased when the women consumed the HSAFA-diet compared with the HUFA-diet (P = 0.02). Plasma t-PA antigen was correlated with plasminogen activator inhibitor type 1 (PAI-1) activity when the participants consumed all three diets (Rs = 0.78, P < 0.01; Rs = 0.76, P < 0.01; Rs = 0.66, P = 0.03; on the HSAFA-, the LSAFA- and the HUFA-diet, respectively), although the diets did not affect the PAI-1 levels. There were no significant differences in postprandial variations in t-PA activity, factor VII coagulant activity or fibrinogen levels due to the diets. Serum fasting Lp(a) levels were lower when women consumed the HSAFA-diet (13%, P < 0.001) and tended to be lower when they consumed the LSAFA-diet (5.3%, P = 0.052) than when they consumed the HUFA-diet. Serum Lp(a) concentrations did not differ when the women consumed the HSAFA- and LSAFA-diets. In conclusion, our results indicate that a coconut oil-based diet (HSAFA-diet) lowers postprandial t-PA antigen concentration, and this may favorably affect the fibrinolytic system and the Lp(a) concentration compared with the HUFA-diet. The proportions of dietary saturated fatty acids more than the percentage of saturated fat energy seem to have a beneficial influence on Lp(a) levels.

A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria.

Transport of preproteins into the mitochondrial matrix is mediated by the presequence translocase-associated motor (PAM). Three essential subunits of the motor are known: mitochondrial Hsp70 (mtHsp70); the peripheral membrane protein Tim44; and the nucleotide exchange factor Mge1. We have identified the fourth essential subunit of the PAM, an essential inner membrane protein of 18 kD with a J-domain that stimulates the ATPase activity of mtHsp70. The novel J-protein (encoded by PAM18/YLR008c/TIM14) is required for the interaction of mtHsp70 with Tim44 and protein translocation into the matrix. We conclude that the reaction cycle of the PAM of mitochondria involves an essential J-protein.

Effects of dietary fat quantity and composition on fasting and postprandial levels of coagulation factor VII and serum choline-containing phospholipids.

Dietary fat influences plasma levels of coagulation factor VII (FVII) and serum phospholipids (PL). It is, however, unknown if the fat-mediated changes in FVII are linked to PL. The present study aimed to investigate the effects of dietary fat on fasting and postprandial levels of activated FVII (FVIIa), FVII coagulant activity (FVIIc), FVII protein (FVIIag) and choline-containing PL (PC). In a randomized single-blinded crossover-designed study a high-fat diet (HSAFA), a low-fat diet (LSAFA), both rich in saturated fatty acids, and a high-fat diet rich in unsaturated fatty acids (HUFA) were consumed for 3 weeks. Twenty-five healthy females, in which postprandial responses were studied in a subset of twelve, were included. The HSAFA diet resulted in higher levels of fasting FVIIa and PC compared with the LSAFA and the HUFA diets (all comparisons P< or =0.01). The fasting PC levels after the LSAFA diet were also higher than after the HUFA diet (P<0.001). Postprandial levels of FVIIa and PC were highest on the HSAFA diet and different from LSAFA and HUFA (all comparisons P< or =0.05). Postprandial FVIIa was higher on the HUFA compared with the LSAFA diet (P<0.03), whereas the HUFA diet resulted in lower postprandial levels of PC than the LSAFA diet (P<0.001). Significant correlations between fasting levels of PC and FVIIc were found on all diets, whereas FVIIag was correlated to PC on the HSAFA and HUFA diet. The present results indicate that dietary fat, both quality and quantity, influences fasting and postprandial levels of FVIIa and PC. Although significant associations between fasting FVII and PC levels were found, our results do not support the assumption that postprandial FVII activation is linked to serum PC.

The serum LDL/HDL cholesterol ratio is influenced more favorably by exchanging saturated with unsaturated fat than by reducing saturated fat in the diet of women.

We compared the effects of a high fat diet [38.4% of energy (E%) from fat; HSAFA diet, polyunsaturated/saturated fatty acid (P/S) ratio = 0.14], a low fat diet (19.7 E% from fat; LSAFA diet, P/S = 0.17), both based on coconut oil, and a diet with a high content of mono- and polyunsaturated fatty acids (PUFA; 38.2 E% from fat; HUFA diet, P/S = 1.9) on serum lipoproteins. The 25 women studied consumed each diet for 3-wk periods in a crossover design. The two high fat diets were identical except for the quality of the test fat. The LSAFA diet was identical to the HSAFA diet except that half the fat was replaced by carbohydrates. Serum total cholesterol, LDL cholesterol and apoB concentrations did not differ between the HSAFA and the LSAFA diet periods. Total cholesterol, LDL cholesterol and apoB were lower when women consumed the HUFA diet than when they consumed the other two diets. HDL cholesterol and apoA-I were 15 and 11%, respectively, higher when women consumed the HSAFA diet than when they consumed the LSAFA diet; HDL cholesterol and apoA-I were lower when women consumed the HUFA diet than when they consumed the HSAFA diet, but not the LSAFA diet. The LDL cholesterol/HDL cholesterol and apoB/apoA-I ratios were higher when women consumed the LSAFA diet than when they consumed the HSAFA diet. The LDL/HDL cholesterol ratio was higher when women consumed either the LSAFA or the HSAFA diet than when they consumed the HUFA diet, whereas apoB/apoA-I was higher when women consumed the LSAFA diet than when they consumed the HUFA diet. Triacylglycerol and VLDL cholesterol were higher when women consumed the LSAFA diet than when they consumed either the HSAFA or the HUFA diet. We conclude that, to influence the LDL/HDL cholesterol ratio, changing the proportions of dietary fatty acids may be more important than restricting the percentage of total or saturated fat energy, at least when derived mainly from lauric and myristic acids, both of which increase HDL cholesterol.

Mitochondrial import of the ADP/ATP carrier: the essential TIM complex of the intermembrane space is required for precursor release from the TOM complex.

The mitochondrial intermembrane space contains a protein complex essential for cell viability, the Tim9-Tim10 complex. This complex is required for the import of hydrophobic membrane proteins, such as the ADP/ATP carrier (AAC), into the inner membrane. Different views exist about the role played by the Tim9-Tim10 complex in translocation of the AAC precursor across the outer membrane. For this report we have generated a new tim10 yeast mutant that leads to a strong defect in AAC import into mitochondria. Thereby, for the first time, authentic AAC is stably arrested in the translocase complex of the outer membrane (TOM), as shown by antibody shift blue native electrophoresis. Surprisingly, AAC is still associated with the receptors Tom70 and Tom20 when the function of Tim10 is impaired. The nonessential Tim8-Tim13 complex of the intermembrane space is not involved in the transfer of AAC across the outer membrane. These results define a two-step mechanism for translocation of AAC across the outer membrane. The initial insertion of AAC into the import channel is independent of the function of Tim9-Tim10; however, completion of translocation across the outer membrane, including release from the TOM complex, requires a functional Tim9-Tim10 complex.

Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel.

The protein insertion complex of the mitochondrial inner membrane is crucial for import of the numerous multitopic membrane proteins with internal targeting signals. Little is known about the molecular mechanism of this complex, including whether it forms a real channel or merely acts as scaffold for protein insertion. We report the unexpected observation that Tim22 is the only essential membrane-integrated subunit of the complex. Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters. The channel is voltage-activated and specifically responds to an internal targeting signal, but not to presequences. Thus, a protein insertion complex can combine three essential functions, signal recognition, channel formation, and energy transduction, in one central component.